Role of Intestinal Microbiota in Ulcerative Colitis

More documents

Recommendations

Info

[60], colonic tissue [46] or cell lines [31]. However, they often only provide short‐term information regarding axenic cultures and tend to ignore the microbial interaction between and within the luminal and mucosal microbiota. Only recently, a dynamic in vitro gut model has been developed which simulates both the luminal and mucosal environment [61]. This model, named the M‐SHIME was adapted from the validated Simulator of the Human Intestinal Microbial Ecosystem (SHIME) [59], and hence allows studying the mucosal microbiota and interaction between luminal and mucosal microbial communities. The aim of this study was to investigate the ability of fecal microbiota from healthy subjects and UC patients in either remission or relapse to colonize the artificial mucus layer of the M‐SHIME. Denaturing Gradient Gel Electrophoresis (DGGE) and quantitative Real‐Time PCR (qPCR), both of which are culture‐independent methods, were applied. DGGE was used to obtain fingerprints of the total microbiota of the three groups after colonization in the M‐SHIME, while qPCR was used to quantify a broad range of selected bacteria taxa presumed to play a role in the homeostasis of the colonic microbial ecosystem. Materials and Methods Human volunteers and clinical characteristics of the UC patients Fecal samples were obtained from 8 patients with UC and 4 healthy controls. Within the UC group, 4 patients were in clinical remission and 4 patients had active disease at the time of sampling according to clinical and endoscopical criteria [6]. The patients were previously diagnosed with UC according to standardized diagnostic criteria at the Department of Gastroenterology, Herlev Hospital [30]. The study was performed in accordance with the Second Helsinki Declaration, reported to the Danish Data Protection Agency and approved by the Regional Ethics Committee. Written, informed consent was obtained from each participant under a protocol approved by the Danish National Committee on Biomedical Research Ethics. UC patients received mesalazine (anti‐ inflammatory drug) and azathioprine (immunosuppressive drug). One of the patients had active pancolitis, one other had active left‐sided colitis and the rest had either active proctitis or proctosigmoiditis. None of the participants had been treated with antibiotics for at least 2 months 4
efore enrolment and there was no significant difference (P = 0.32) in the mean age of the participants comparing the 3 groups. Sample collection and processing Stool samples were collected at the home of the participant in airtight containers and stored at 4°C (limited storage time was encouraged [44]) until delivery to the laboratory, where they were processed immediately. Feces were homogenized in glycerol to give a 25% feces/glycerol slurry. This was performed in an anaerobic cabinet (Macs Work Station, Don Whitley, containing 10% H2, 10% CO2, and 80% N2). The processed samples were stored at ‐80°C until further analysis. Growth medium and chemicals Unless stated otherwise, chemicals were obtained from Sigma (Bornem, Belgium). The M‐SHIME feed contained 1.0 g/l arabinogalactan, 2.0 g/l pectin, 1.0 g/l xylan, 3.0 g/l starch, 0.4 g/l glucose, 3.0 g/l yeast extract, 1.0 g/l peptone, 4.0 g/l mucin, and 0.5 g/l cystein. Pancreatic juice contained 12.5 g/l NaHCO3, 6.0 g/l bile salts (Difco, Bierbeek, Belgium) and 0.9 g/l pancreatin. Mucin agar was prepared by boiling autoclaved distilled H2O containing 5% porcine mucin type II and 1% agar. The pH was adjusted to 6.8 with 10 M NaOH. M‐SHIME A dynamic in vitro model for the human intestinal tract, which accounts for both the luminal and mucosal microbiota, was designed by Van den Abbeele et al. [61]. The M‐SHIME set up consisted of two vessels simulating the stomach and the small intestine, and six ascending colon vessels, which were run in parallel without the transverse and descending colon (Figure 1). All ascending colon vessels were modified by incorporating mucin‐covered microsms into the luminal suspension. 80 mucin‐covered microcosms were added per 500 ml luminal suspension. The microcosms (length = 7 mm, diameter = 9 mm, total surface area = 800 m2/m3, AnoxKaldnes K1 carrier, AnoxKaldnes AB, Lund, Sweden) were coated by submerging them in mucin agar. Each ascending compartment (500 ml) was inoculated with 27 ml of a 1:10 dilution of the feces/glycerol slurry. Inoculum preparation was done as described previously [48]. Three times per day, 140 ml SHIME feed and 60 ml pancreatic juice were added to the stomach and small intestine, 5
Page 1:
Role of Intestinal Microbiota in Ul
Page 4 and 5:
Role of Intestinal Microbiota in Ul
Page 6 and 7:
Preface Preface This thesis present
Page 8 and 9:
Summary Summary The microbiota of t
Page 10 and 11:
Dansk sammendrag Dansk sammendrag M
Page 12 and 13:
Introduction and objectives Introdu
Page 14 and 15:
List of Manuscripts Not included in
Page 16 and 17:
List of contents List of Centents P
Page 18 and 19:
List of Centents Methodology append
Page 21 and 22:
1. The intestinal environment Theor
Page 23 and 24:
Theoretical part 5 1. The intestina
Page 25 and 26:
2. The colonic environment Theoreti
Page 27 and 28:
Theoretical part 9 2. The colonic e
Page 29 and 30:
Table 1: The presence of glycoside
Page 31 and 32:
Theoretical part Figure 3: The colo
Page 33 and 34:
3. Inflammatory Bowel disease Theor
Page 35 and 36:
Theoretical part 17 3. Inflammatory
Page 37 and 38:
Theoretical part 19 4. Modulation o
Page 39 and 40:
Theoretical part 21 4. Modulation o
Page 41 and 42: Theoretical part 23 4. Modulation o
Page 43 and 44: Table 4: Clinical trials on the pre
Page 45 and 46: Theoretical part 5. Production of p
Page 51: Methodology part
Page 54 and 55: Methodology part 6. Methodology, co
Page 60 and 61: Introduction Methodology part 42 Pa
Page 62 and 63: Abstract Background Detailed knowle
Page 64 and 65: depending the level of disease acti
Page 66 and 67: in 1 x TAE at 60 °C for 16 h at 36
Page 68 and 69: Statistics PCA were generated by SA
Page 70 and 71: The PCA of the Gram‐positive bact
Page 72 and 73: layer of UC patients and found that
Page 74 and 75: Acknowledgements The authors thank
Page 76 and 77: Table 2 ‐ 16S rRNA gene and 16S
Page 78 and 79: 1. Firmicutes phylum 2. Bacteroidet
Page 80 and 81: Supplementary Figure S1. Dice clust
Page 82 and 83: Reference List 1. Ahmed S, Macfarla
Page 84 and 85: 32. Matsuki T, Watanabe K, Fujimoto
Page 87 and 88: Methodology part Paper 2 Fecal lact
Page 89 and 90: Fecal lactobacilli and bifidobacter
Page 91: Introduction The mucus layer lining
Page 95 and 96: (Bio‐Rad Labs, Hercules, Californ
Page 97 and 98: Microbial community analysis using
Page 99 and 100: difference from the luminal microbi
Page 101 and 102: that C. coccoides group and C. lept
Page 103 and 104: Table 1 ‐ 16S rRNA gene of phylum
Page 105 and 106: Table 2 ‐ Preference of bacterial
Page 107 and 108: Figure 1. A) Schematic overview of
Page 109 and 110: A. B. Figure 3. Principal component
Page 111 and 112: 15. Fooks LJ, Gibson GR. (2002) In
Page 113 and 114: 47. Ouwehand AC, Suomalainen T, Tol
Page 115 and 116: Methodology part Paper 3 Paper 3 In
Page 117 and 118: APPLIED AND ENVIRONMENTAL MICROBIOL
Page 119 and 120: 8338 VIGSNÆS ET AL. APPL. ENVIRON.
Page 125: 8344 VIGSNÆS ET AL. APPL. ENVIRON.
Page 128 and 129: Methodology part Introduction The a
Page 130 and 131: Journal of Agricultural and Food Ch
Page 139 and 140: Methodology part Paper 5 Paper 5 Ma
Page 141 and 142: Appl Microbiol Biotechnol (2011) 90
Page 143 and 144:
Appl Microbiol Biotechnol (2011) 90
Page 145 and 146:
Page 147 and 148:
Page 149 and 150:
Page 151 and 152:
Page 153 and 154:
Methodology part Paper 6 Tailored e
Page 155 and 156:
Process Biochemistry 46 (2011) 1039
Page 157 and 158:
Table 1 List of enzymes. Enzyme Sou
Page 159 and 160:
J. Holck et al. / Process Biochemis
Page 161 and 162:
Intensity 50 0 C1 175.05 J. Holck e
Page 163 and 164:
J. Holck et al. / Process Biochemis
Page 165:
[29] Bauer S, Vasu P, Persson S, Mo
Page 168 and 169:
Methodology part 150
Page 170 and 171:
Methodology part 152 Appendix 1 hor
Page 172 and 173:
Appendix 3 Methodology part Appendi
Page 174 and 175:
Methodology part 156 Appendix 3
Page 177 and 178:
7. Discussion and perspectives Disc
Page 179 and 180:
Discussion and conclusion 161 7. Di
Page 181 and 182:
Page 183 and 184:
Page 185 and 186:
References References Abreu,M.T., V
Page 187 and 188:
References Derrien,M., Vaughan,E.E.
Page 189 and 190:
References Haarman,M. and Knol,J. (
Page 191 and 192:
References Lantz,P.G., Matsson,M.,
Page 193 and 194:
References Matsuki,T., Watanabe,K.,
Page 195 and 196:
References Pullan,R.D., Thomas,G.A.
Page 197 and 198:
References Tannock,G.W. (2010). Ana
Page 200:
National Food Institute Technical U
show all

Role of Intestinal Microbiota in Ulcerative Colitis

Create successful ePaper yourself

Delete template?

Save as template?